Meeting the Challenges of Bioanalysis...Key Challenges in Routine Bioanalysis Operation Sensitivity...

©2009 Waters Corporation 1

Meeting the Challenges of Bioanalysis

Impact of Recent Advances in UPLC and Mass Spectrometry

Dr. Jing Lin


Outline

Introduction

Meeting the key challenges in bioanalysis

—Increasing sensitivity

—Enhancing specificity

—Improving productivity

Summary


Introduction

Bioanalysis plays an important role in drug development

Quantitative analysis of compounds and metabolites in

biological matrices is normally achieved by LC/MS/MS or

GC/MS(MS)

Bioanalysis forms part of the regulatory submissions for new

drug application, IND etc. in GLP studies

Bioanalytical laboratories are facing many common challenges

— Increasingly stringent regulations

— Higher business pressure and tougher competition

— New and evolving technical/scientific challenges

©2009 Waters Corporation Company Confidential 4

Matrix Effects

New guidance on the determination and acceptability of matrix

effects as a result of sample preparation

Should be assessed by comparison in six different sources of

matrix if using analog internal standard (IS)

— Variability (%CV) across 6 lots


Incurred Sample Reanalysis (ISR)

Require a % of patient samples be re-analyzed and compared to

original

— 5% to 10% of the study samples

Various acceptance criteria were discussed and a consensus

proposed

— 4/6/20 rule‗: 2/3 of the reanalysed samples have to show

< 20% variation of the original (?) value

Must have SOP for ISR in place

Direct impact on productivity and cost of analysis

Regulatory Changes in Bioanalysis (2)

©2009 Waters Corporation | COMPANY CONFIDENTIAL 6

Regulatory Changes in Bioanalysis (3)

Metabolite In Safety Testing (MIST)

Newest guideline published in June 2009

— ICH Topic M 3 (R2)

— http://www.emea.europa.eu/pdfs/human/ich/028695en.pdf

Simplified and less stringent guideline than initially discussed

Nonclinical characterization of a human metabolite(s) is only

warranted when that metabolite(s) is observed at exposures

greater than 10% of total drug-related exposure and at

significantly greater levels in humans than the maximum

exposure seen in the toxicity studies.

Such studies should be conducted to support Phase III clinical

trials.

http://www.emea.europa.eu/pdfs/human/ich/028695en.pdf


Key Challenges in Routine Bioanalysis Operation

Sensitivity

— Ever increasing demand

— New highly potent drugs, low level of metabolites

— Innovative formulation: controlled-release, inhalation…

— Specific population studies: pediatric …

— Implementation of Dried Blood Spot

Specificity

— Monitoring & Minimizing matrix interference: specific FDA guideline

— Detecting and quantifying metabolites: MIST guideline

Productivity

— Analyze more samples

— Maximizing system up-time

— Easy access to non specialist users

— Efficient workflow


Xevo QTof


Increasing The Sensitivity


Optimizing ESI Desolvation

High temperature heater

— High temperature alloy

element

— 650oC required for optimal ESI

operation

Optimal zone of desolvation

— Balanced gas distribution

— Vertical nebulizer positioning


Optimizing Gas Flow Dynamics

Shape of chamber

— Smooth, curved surface

— Reduces turbulence

— Excellent beam stability

Size of chamber

— Efficient desolvation

— Excellent linear response

Temperature of chamber

— Thermally regulated

— Prevents solvent deposition

Tangential exhaust

— Gas flows modeled to efficiently

remove non-ionized materials from

source chamber


Meeting the Sensitivity Challenge with Xevo™ TQ MS

Fluticasone Dipropionate is a

corticosteroid used in the

treatment of asthma

Inhaled delivery results in low

circulating plasma levels

Assay requirements are for

low pg/mL sensitivity

Xevo TQ MS delivers pg/mL

sensitivity

Fluticasone Propionate 3 pg/mL in Plasma

O

OH

COSCHF

OCOCH

Time1.10 1.20 1.30 1.40 1.50 1.60

%

0

100

090309_HLB_QUAN_029 Sm (Mn, 2x1) MRM of 3 Channels ES+ 501.3 > 293.2 (Fluticasone)

3.76e3

1.19

1.15

1.25

1.23

1.561.33


Improving Assay Sensitivity with UPLC®

Assay sensitivity is dependent upon peak intensity

Extra efficiency of UPLC® produces sharper,

narrower peaks

Higher linear velocities of UPLC and shorter

columns also give sharper peaks

UPLC = Faster, sharper, more intense peaks


5-fold increase in sensitivity as

a result of UPLC

UPLC analysis allowed the

quantification of the 24 hr

samples

New data indicated a different

elimination phase of the

pharmacokinetics significantly

affecting the oral bioavailability

Samuele Pedraglio ∗, Marco Giulio Rozio, Paola Misiano,Veronica Reali, Giulio Dondio, Chiara Bigogno; J Pharm. Biomed Anal 44 (2007) 665–673

The impact of UPLC and raised sensitivity on a PK profile

Solving Bioanalytical Challenges with

UPLC® Technology: Sensitivity


Enhancing The Specificity

- Minimizing matrix effect

- Confirming peak identity

- Detecting & Quantifying metabolites


Separation of

metabolite

critical pairs is

essential for accurate

quantitation and is

becoming more

important in order to

satisfy increasing

Regulatory

expectations

(MIST)

Ref: Donegan M,BMS, www.cosmoscience.org/archive_2008.htm

Impact of UPLC® on Resolution


Simple (but dirty) sample preparation by protein

precipitation (PPT)

Phospholipids are the main remaining matrices

after sample clean-up

Evaluate impact of UPLC on matrix effect vs

HPLC

Quantification Of TerfenadineIn Human Plasma


0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

%

0

MRM of 2 Channels ES+

2.17e4

Terfenadine

0.50 1.00 1.500

472.1 > 436.1

1.37e4

1.09S/N = 81PhospholipidsTerfenadine

0.1 ng/mL Terfenadine

PPT

High pH

S/N 81

>30% Suppression

Resolution and Sensitivity:HPLC, High pH


0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

%

0

MRM of 2 Channels ES+

2.17e4

Terfenadine

0.50 1.00 1.500

472.1 > 436.1

1.37e4

1.09

S/N= 482

Minutes

PhospholipidsTerfenadine

0.1 ng/mL Terfenadine

PPT

High pH

S/N 482 (6X HPLC)


Are matrix effects measured in HPLC and UPLC the same or

statistically different?

10 analytes run on both HPLC and UPLC

— With different pHs, gradients and sample concentrations

Data were paired according to identical conditions

—An HPLC data point with a UPLC data point under the same

conditions

43 sets of paired data subjected to paired t-test

A p value less than 0.05 indicates 2 populations are different

Paired t-test returned a ―p‖ value of 0.0006

—There is a difference between matrix effects measured in HPLC

versus UPLC™

Therefore, the reduction in matrix effects with UPLC™ is

statistically significant

Statistical Comparison of Matrix Effects


Assessment of Matrix Effects and Metabolites with MS

Require acquisition of qualitative (spectral) data

Targeted and untargeted approaches can be used

Parent ion scan for detection of targeted/expected

matrix/metabolites

— Detection of lipid interference or metabolites with common

fragment ions

Full-scan MS for detection of untargeted /unexpected

matrix/metabolites

MS/MS spectra for confirmation of peak identity


Conventional Tandem Quadrupole vsXevo TQ MS

Standard tandem quadrupole MS

— Very sensitive in MRM mode

— Significantly less sensitive when acquiring in spectral mode

o Full scan MS, MS/MS or parent ion scan

— Switching between MS and MS/MS is nearly impossible

Xevo TQ MS

— Provides outstanding MRM sensitivity

— Incorporates ScanWave collision cell technology

o Innovative way of using existing travelling wave technology

— Significantly enhances sensitivity in spectral mode

— Designed for rapid MS to MS/MS switching

— Collects more useful information from a single experiment

o High quality Quantitative & Qualitative data simultaneously


Standard Product Ion Scanning

Continuous output of ions by cell

Most ions filtered out by MS2

Very inefficient


ScanWave EnhancedProduct Ion Scanning

Ion accumulation

Mass selective ion ejection

Synchronised with scanning of MS2


ScanWave Enhanced sensitivity full scan MS/MS

Travelling Wave

DC Barrier RF BarrierPotential Energy

Low m/z Ion

High m/z Ion

Intermediate m/z Ion

Storage Region ScanWave Region

Travelling Wave

Travelling Wave

Accumulation

Transfer

High m/z Ejection

Medium m/z Ejection


Standard

Data

ScanWave

Enhanced

Data

ScanWave …enhanced sensitivity full scan MS/MS

Scan speed = 5,000 amu/second

Conventional

product ion spectrum

ScanWave enhanced

product ion spectrum


PIC Scan

Acquisition

MRM

Acquisition

PIC SCAN:

MRM data is used as a specific

trigger for the acquisition of a

ScanWave enhanced product

ion spectrum.

Rapid Mode Switching Product Ion Confirmation Scan


UPLC/MS/MS Analysis of Fluticasone Propionate in Plasma

MRM data

Which peak is the

Fluticasone?


PICSComparison in TargetLynx

Reference Spectrum from Quanpedia


Dual Scan-MRM

How does it work?

Rapidly alternates between MRM and

MS full scan acquisition modes.

5ms switch time

No compromise to MRM or MS full scan

How is MS data acquired on Xevo TQ?

Collision cell remains pressurised in both modes

MS2 scans to produce fast MS spectra

T-wave ensures MS ions transit efficiently.


Dual Scan-MRM

15 points Across1.5 Sec Peak

Full Scan MS

(Matrix)

MRM

(Analyte)

Excellent MRM peak definition

+

Full Scan data acquisition

Faster LC/MS/MS method

development and matrix

monitoring

Simpler detection and

confirmation of active

compounds and associated

metabolites.


Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

%

0

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

%

0

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

%

0

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

%

3

220109_SW_DS_133 5: MS2 ES- TIC

1.72e9

2.31;282.81.77;278.90.33;400.7 1.48

168.81.42;488.7

1.21;523.40.96;748.6

2.17;885.4 2.46;851.3

2.68;792.0 3.03791.9

3.19833.3

220109_SW_DS_133 4: Parents of 184ES+ TIC

7.54e9

2.54;757.52.15519.5

1.98541.4

0.13757.8

2.26;523.2

220109_SW_DS_133 3: Parents of 227ES+ TIC

1.26e7

1.55;324.3

0.17348.6

0.67270.80.26;270.3

1.44294.1

1.19320.3

0.98294.4

0.79306.0

1.69;326.0 2.24242.0

1.97348.0

2.12226.2

2.36241.1

2.52269.0

3.13296.0

2.70225.1

3.07;286.6 3.19;331.8 3.71293.9

3.62;225.3

220109_SW_DS_133 2: MS2 ES+ TIC

1.15e10

2.16;183.61.41;125.50.26119.7

1.98181.0

1.61313.7

2.39;148.5 2.54;758.4

3.03369.0

3.32180.6

220109_SW_DS_133 1: MRM of 3 Channels ES+ 325 > 297 (Alprazolam Metabolite)

7.86e6

1.56;325.0

220109_SW_DS_133 1: MRM of 3 Channels ES+ 314.2 > 286.1 (AlprazolamD5)

5.18e6

1.60;309.2

220109_SW_DS_133 1: MRM of 3 Channels ES+ 309.2 > 281 (Alprazolam)

8.32e6

1.60;309.2

Quantify MRM

Quantify MRM of Internal Std

Quantify MRM of Metabolite

Matrix Full Scan +ve

Monitor Analyte Common Fragment

Monitor Lipid Common Fragment

PIC Spectrum

Matrix Full Scan -ve

Detect, Quantify, Confirm All in One Run


Improving The Productivity

- Reducing analysis time

- Maximizing system up-time

- Easy access

- Efficient workflow


Reducing Analysis Time with ACQUITY UPLC

HPLC-MS/MS

%

Time2.0 2.5 3.01.50.5 1.00

UPLC-MS/MS

Reduced run timeMore sensitive Better resolution

To benefit from the advantages of UPLC the MS must be designed to be fully compatible


MS Acquisition Speed

The MS must be capable of acquiring at data rates

compatible with narrow UPLC peaks without loss in

performance

Good quality quantification requires:

— 10-15 MRM data points per chromatographic peak

— No loss in signal

— No increase in cross talk

Latest MS design provides

— 6ms per MRM data point (167 data points per second)

— 20 ms to switch between + and – modes

— 20 ms to switch between ESI and APCI (ESCi)


T-Wave For Speed

Traditional hexapolereplaced with

Stacked ring electrodes

T-Wave used to control the transit time of ionsRapidly clearing between channels

•(eliminates cross talk)Rapidly refilling

•(no signal loss)


Engineered Simplicity

Best performance can only be

routinely achieved if the

instrument is designed to be

simple to operate and service

Innovative ion source

IntelliStart (ready to run)

technology


New API Source DesignPerformance, Usability & Serviceability

ZSpray™

— A time proven geometry

(many years of experience

& 1000s in use daily)

— Robust performance in the face of

complex biological samples

Designed to be compatible with

high flow HPLC and UPLC®

applications

Innovative, Ergonomic Engineering

— Tool free maintenance

— Novel tool free source exchange

— Integrated gases

— Plug and play probes


Integrated UPLC/MS by Design

Column Optimally

positioned to eliminate

band broadening

Built-in Fluidics

Automated delivery of

reference solutions

Automated flow

switching


IntelliStart

Automated system

calibration

Automated sample

tuning and method

development

Automated, pre-

analysis system

performance check


XXSystem Suitability Check

LC/MS System check: Run 1

LC/MS System check: Run 6

%

0

100

Time

%

0

100

Time

Peak area = 4351.6

Peak area = 4435.2

LC/MS System Check

• System suitability check automatically queued

• Instrument Pass/Fail calculated based on user-defined parameters

• Results stored to ELN database, or email to supervisor

• Instrument status displayed on instrument interface


Optimization of MS Conditions

Line from infusion

pump, contains

analyte

Line from LC pump,

contains mobile phase

Probe may need to

be changed

Infusion Setup Xevo Setup


Developing an MRM Method:Automated Parent & Fragment Optimization

Step 1

IntelliStart™

— Input the compound

name

— Input the parent

mass or formula

— Select the adduct

— Click Start and get a

coffee


Developing an MRM Method:Reporting

IntelliStart™ Results Reporting:

Ionization mode

Best MRM transition(s)

Cone voltages and Collision energies

Generation of a report showing how parameters were selected

All parameters automatically populated into the method set


Comparison of Manual vs. IntelliStart MRM Method Development: 5 Analytes

Manual Development Time IntelliStart™ and ESCi®

Plumb for infusion(time taken to find all the bits needed….)

2-5 min Enter file name, mass or formula and adduct into IntelliStartPress StartCompound is automatically infused

Change probes for different ionization

2 min ESCi does this automatically

Check cone voltages 2 min IntelliStart does this automatically

Check collision energies

2 min IntelliStart does this automatically

Optimize gas flow 2 min IntelliStart does this automatically

Optimize temperature 2 min IntelliStart does this automatically

Adjust parameters for optimum conditions

2 min IntelliStart does this automatically

Total time:15 min X 5 = at least 75 mins

Total time (while you were away…..)4 min X 5 = 20 min, AUTOMATED


Select compounds

from database

Ready to

Acquire &

Process

Create

LC/MS

methods

Use Predefined Analysis

Use Predefined Compounds

QuanPedia™

A central LC/MS/MS data base based

on compound name

Allowing methods to be stored,

controlled, shared, and downloaded

across multiple instruments and sites


QCMonitor: Automated Monitoring of Assay Performance

Real-time, proactive monitoring of:

— Blanks

— Standards

— Quality control

Action on failure:

— Flag error

— Terminate sample run

Prevent repeated analysis

Prevents loss of precious samples

— No injection if the LC/MS system is not performing appropriately.


Data Processing with TargetLynx™

Advanced software package for quantitative analysis

— ApexTrack™ enabling accurate and consistent integration

— A full range of automatic quality control checks

— Simple rapid data review with intuitive browser

— Data re-processing directly from the browser

Fully compliant data processing

— Full audit trail recorded for data processing activities

— Electronic signature and secure data management tools

Data export function

— Allows for generation of partial or full sets of digital information, in

ASCII or XML, for import into other systems including statistical

analysis packages and LIMS software

http://www.waters.com/watersdivision/images/products/3quan.htm


TrendPlot

A tool to monitor long-term data

trends/system performance

Monitor LOD/LOQ performance

Track internal standard response

Track QC response

Plot intra- and inter-batch data

from TargetLynx


Setup instrumentation

Create LC/MS/MS methods

Monitor data proactively

Process, qualify & report

Plot trends in results

Efficient Bioanalysis Workflow

IntelliStart™

QuanPedia™

QCMonitor™

TargetLynx™

TrendPlot


Summary


Impact of Recent Advances in UPLC & MS Technology

Increasing Sensitivity

— Contribution of UPLC is real and proven

— New source design allowing more efficient ionization

Enhancing Specificity

— UPLC providing better resolution and minimizing co-elutions

— ScanWave increasing available acquisition options, allowing

quantification, confirmation and detection of drug and

metabolites simultaneously

Improving Productivity

— UPLC reducing analysis time

— Fully integrated UPLC MS system designs

— Engineered simplicity

— Efficient workflow : from method development to data processing


Acknowledgements

Erin Chambers

Diane Diehl

Rob Plumb

Joanne Mather

Paul Rainville

Geneen Baynham

Jessalynn Wheaton

David Little

Gary Harland

Meeting the Challenges of Bioanalysis...Key Challenges in Routine Bioanalysis Operation Sensitivity...

Documents

Transcript of Meeting the Challenges of Bioanalysis...Key Challenges in Routine Bioanalysis Operation Sensitivity...